1. Field of the Invention
The present invention concerns a bioreactor device and computer methods of utilizing the device to monitor, analyze and control the course of growth of cells. Further, the invention concerns automated flexible modification of control parameters during the growth of the cells based on data obtained from sensors. The data discloses both continuous and transient growth effects. The modifications during growth can determine optimum conditions for growth or, conversely, conditions limiting growth.
2. Description of the Background
Prediction of the course of cellular growth in fermentations (biogas, biomass, bioconversions, bioremediation, foods/feed, pharmaceuticals/medicines) is a complex process requiring knowledge and investigation of many basic physiologic parameters. Historically, the effect of physiologic parameters such as temperature, salinity, nutrients, cofactors, inhibitors, or pH may involve independent measurement. For example, normally an investigator places a series of individual cultures in incubators set at a series of temperatures. Investigation of multiple parameters and their interactions may involve a multivariate approach. The same is true of the other parameters. In the strict sense, these experiments only determine the “best” initial condition. They do not actually measure the graded effect of change during growth.
Much of traditional microbial or cellular physiology involved elucidating the individual steps in the metabolism of a substrate and combining these steps into a pathway. Isotope tracing and genetic mutation were used to determine if the pathway actually worked. Investigations of the physiology of systems under growing conditions were largely ballistic in design, i.e., once the fermentation was started with a set of initial parameters, no changes were made and the results landed where they might. This was true of both closed systems and to a lesser extent to flow systems such as chemostats. Even today, most fermenters or bioreactors sold as having computer control are essentially analog or digital set point devices with the computer able to operate the controls. Desired changes after growth initiation may involve operator intervention. The present state of fermentation art largely involves empirical study of the effects of varying a single parameter during the course of fermentation.
The current state of the art in determining the course of cellular growth is to measure a single parameter such as optical density at fixed wavelength, capacitance measurements, and production of acid, redox potential, radio-frequency dielectrics, luminescence or fluorescence. Where optical techniques are used, the wavelengths are limited to a narrow band relative to the complete spectrum.
A number of commercial instruments utilize such single parametric measures such as:
The Aber Instruments Limited Model 220 biomass monitor which uses radio-frequency dielectrics.
The FOGALE Nanotech Company BIOMASS SYSTEM® is an on-line measurement instrument for determination of viable cell concentration using capacitance technology.
The Turner Designs algaewatch is an on-line monitor that detects algal biomass through chlorophyll fluorescence.
The Sartorius BBI Systems GmbH (BBI) (former B. Braun Biotech International GmbH) FUNDALUXr II system is an absorption-based photometric probe, designed for use in bioreactors and fermentors. The system uses a probe inside the culture vessel, which operates according to the transmittance principle with a wave length in the near infrared (NIR) range.
The optek-Danulat, Inc. Real-time biomass concentration probes use near infrared absorption. The series of bioprocess analyzers were designed specifically to integrate easily into existing bioreactors and fermenters.
All of the above instruments can monitor cell concentration and have some computational capacity. None of them use the information gathered in a control loop to modify the course of the fermentation.
U.S. Pat. No. 6,673,532 to Rao, uses non-invasive optical chemical sensing technology wherein an optical excitation source excites an optical chemical sensor. This system relies on single excitation wavelength bands and specific chemical sensor bands to monitor growth in small parallel vessels (e.g., 96 well plates).
With the exception of the system described by Rao, all the above instruments utilize a probe inserted in the fermentation vessel. The first use of such a probe was described in 1971 (Robrish, S. A., LeRoy, A. F., Chassy, B. M., Wilson, J. J., and Krichevsky, M. I., Use of a fiber optic probe for spectral measurements and the continuous recording of the turbidity of growing microbial cultures. Appl. Microbiol., 21: 278-287 {1971}). Later, a prototype autoclavable light probe to measure the concentration of bacterial mass was developed. In the 1971 paper, the simultaneous production of acid and the increase in biomass by a homolactic streptococcus was demonstrated.
In the above examples, the information produced by the probes or chemical sensors are usable for the operator to change parameters. None provide computer algorithms to automatically direct changes towards specific physiologic goals.